Study On The Purification And Properties Of Lectin From The Pleurotus Ferulae Lenzi

The purpose of this study was to extract and purificate one lectin from the Pleurotusferulae and mainly study the basic physical and chemical properties of lectins and partimmunomodulatory activity were obtained for build a solid foundation at the further PFL’sexploration in the in theory.1Lectins separation and purification from the Pleurotus ferulae.The highly purified lectin was recived by PBS (phosphate buffer saline) extraction offruit body mash, salting out with ammonium sulfate, ultrafiltration and concentration,subjected to ion-exchange chromatography on DEAE-cellulose followed by affinitychromatography on sepharose-4B. Select chromatography column is36cm long and1.1cmoutside diameter; choose3：1as column material and packing ratio in DEAE-celluloseion-exchange chromatography, Sepharose-4B activation treated by NaOH is the materialPacking in affinity chromatography and the column material, the packing ratio is1：1.Miscellaneous protein were eluted by PBS equilibrium liquid to A280<0.02and collecteluent to dialysis electrophoresis, shows no obvious protein bands; With0.25mol/LD-galactose desorption to A280<0.02,1mL/min flow rate was limited, collect eluent todialysis electrophoresis and a single protein electrophoresis banding was observed, Thereare indications that PFL as a single dimer composed by a single subunits and confirmed thatthe high purity PFL we obtained.②PFL electrophoresisThe result show a single protein of the molecular weight about17.5kDa, bothSDS-PAGE electrophoresis and Native-PAGE electrophoresis, Then the total proteincontent was measured84.33%by using Coomassie brilliant bluestaining R-250in thestandard curve. Native-PAGE without destroying protein natural conditions and electriccharge bathing in the electric field and medium withe natural protein of its own charg. Onthe one hand, the purity of protein isolated can be identified, on the other hand, it is soconvenient to the protein recycling.③Hemagglutinating activity reserch of PFLThe maximum titer of hemagglutination activity is28, The hemagglutination activiesof PFL was destroyed after treatment with temperature higher than70℃or the pH lowerthan2and higher than10.The qualities of the lectin are stable in the pH4~9. when theSalinity increase, the activity decrease and D-galactose is the best inhibitors of letins,cations have different effects on the hemagglutinating activity of lectin.；Zn2+significantlyenhance the hemagglutination activity.④Immunomudulatory activity of lectin form Pleurotus ferulae To observe the influence of PFL on mouse splenocytes proliferation. differentionsconcentrations of PFL with splenocytes proliferation cells were cultured for48h andpromoting proliferation effect was detected by the method of MTT test optical density. Aportion of the beneficial on mouse murine spleen lymphocyte with lectin content increaseswhen the concentrration is only80μg/mL. NO can be generated when eritonealmacrophages in mice stimulated by the PFL. and the level of NO as an important role inkilling tumor immunity, large amounts of NO were produced just the content of lectin onlyacheived40μg/mL to stimulate macrophages play a vital role in Immunomudulatoryactivity. Which provide certain reference basis for the further immune activity study ofPleurotus ferulae.